Solid compositions of fibrin monomer

ABSTRACT

This invention is directed to solid compositions, for example, lyophilized forms, of fibrin monomer. These compositions are useful wherever fibrin monomer could be employed, e.g., as a surgical sealant to provide hemostasis.

This is a divisional application of U.S. Ser. No. 08/138,674 now U.S.Pat. No. 5,750,657 filed Oct. 18, 1993 which is a continuation-in-partapplication of U.S. Ser. No. 07/958,212 filed Oct. 8, 1992, nowabandoned.

FIELD OF THE INVENTION

The subject invention relates to fibrin sealants. More specifically, thesubject invention relates to the use of a fibrin sealant wherein acomposition comprising fibrin monomer or a composition comprisingnoncrosslinked fibrin is utilized as a component of the fibrin sealant.

BACKGROUND OF THE INVENTION

One mechanism for hemostasis, i.e., prevention of blood loss, of amammal is the formation of a blood clot. Clot formation in humans, i.e.,blood coagulation, occurs by means of a complex cascade of reactionswith the final steps being the conversion of fibrinogen--a monomer--bythrombin, calcium ions and activated factor XIII to form ultimatelycrosslinked fibrin II polymer, which is the fibrin clot.

The formation of crosslinked fibrin II polymer proceeds by thefibrinogen being converted by thrombin to fibrin I monomer, whichspontaneously polymerizes to form fibrin I polymer, which is sometimesreferred to as soluble fibrin I because by treatment by appropriatechemical means the fibrin I polymer can be reconverted to fibrin Imonomer. The fibrin. I polymer is then converted by thrombin to fibrinII polymer, which is sometimes referred to as soluble fibrin II becauseby treatment by appropriate chemical means the fibrin II polymer can beconverted to fibrin II monomer. The fibrin II polymer, under theinfluence of factor XIIIa--known as activated factor XIII--is thencrosslinked to form crosslinked fibrin II, which is the fibrin clot.Factor XIII is activated by thrombin in the presence of calcium ions.Cross-linked fibrin II is sometimes referred to as insoluble fibrin IIbecause it cannot be converted to fibrin II monomer.

It should be noted that thrombin is formed from prothrombin. Prothrombinis converted to thrombin by factor Xa in the presence of calcium andother ancillary substances.

Fibrinogen represents about 2 to 4 grams/liter of the blood plasmaprotein. Fibrinogen is a monomer that consists of three pairs ofdisulfide-linked polypeptide chains designated (Aα)₂, (Bβ)₂, γ₂. "A" and"B" represent the two small aminoterminal peptides, known asfibrinopeptide A and fibrinopeptide B, respectively. The cleavage offibrinopeptides A from fibrinogen in the transformation of fibrinogen bythrombin results in the fibrin I compound and the subsequent cleavage offibrinopeptides B results in the fibrin II compound.

Such cleavage of fibrinopeptides A and B reduces the molecular weight offibrinogen by an extremely small amount, about 6,000 out of 340,000daltons, but exposes the polymerization sites. For a review of themechanisms of blood coagulation and the structure of fibrinogen, see C.M. Jackson, Ann. Rev. Biochem., 49:765-811 (1980) and B. Furie and B. C.Furie, Cell, 53: 505-518 (1988).

A fibrin sealant is a biological adhesive whose effect imitates thefinal stages of coagulation, thereby resulting in a fibrin clot.Conventional fibrin sealants consist of concentrated human fibrinogen,bovine aprotinin and factor XIII, as the first component and bovinethrombin and calcium chloride as the second component. Application isgenerally carried out with a double-barrelled syringe, which permitssimultaneous application of both components to the site where one wantsto form the fibrin clot. Aprotinin is a fibrinolytic inhibitor added topromote stability of fibrin sealants.

The fibrinogen component of the fibrin sealant is prepared from pooledhuman plasma. The fibrinogen can be concentrated from the human plasmaby cryoprecipitation and precipitation using various reagents, e.g.,polyethylene glycol, ether, ethanol, ammonium sulfate or glycine. For anexcellent review of fibrin sealants, see M. Brennan, Blood Reviews,5:240-244 (1991); J. W. Gibble and P. M. Ness, Transfusion, 30:741-747(1990); H. Matras, J. Oral Maxillofac Surg., 43:605-611 (1985) and R.Lerner and N. Binur, J. of Surgical Research, 48:165-181 (1990).

Recently, there has also been an interest in the preparation of fibrinsealants that utilize autologous fibrin. An autologous fibrin sealant isa fibrin sealant wherein the fibrinogen component of the fibrin sealantis extracted from the patient's own blood. The use of an autologousfibrin sealant is preferred because it eliminates the risk oftransmission of blood-transmitted infections, e.g., hepatitis B, non A,non B hepatitis and acquired immune deficiency syndrome (AIDS), thatcould otherwise be present in the fibrinogen component extracted frompooled human plasma. See L. E. Silberstein et al., Transfusion,28:319-321 (1988); K. Laitakari and J. Luotonen, Laryngoscope,99:974-976 (1989) and A. Dresdale et al., The Annal of Thoracic Surgery,40:385-387 (1985).

An infection can be transmitted by a fibrin sealant not only by means ofthe fibrinogen but also by means of the bovine aprotinin and the bovinethrombin component. Bovine thrombin has been known to carry theinfectious agent bovine spongiform encephalitis (BSE) and other virusespathogenic to mammals. Furthermore, bovine thrombin is a potent antigen,which can cause immunological reactions in humans. Thus, the use ofbovine thrombin could result in the recipient of the bovine thrombinbeing adversely affected. See D. M. Taylor, J. of Hospital Infection,18(Supplement A):141-146 (1991), S. B.

Prusiner et al., Cornell vet, 81 No. 2: 85-96 (1991) and D. Matthews, J.Roy. Soc. Health, 3-5 (February 1991).

Accordingly, there is the need for a fibrin sealant that can bedelivered to a patient without the risk of viral contamination or otheradverse affects.

SUMMARY OF THE INVENTION

The subject invention relates to a method for utilizing a fibrin sealantwhich comprises:

(a) contacting a desired site with a composition comprising fibrinmonomer; and

(b) converting said fibrin monomer to a fibrin polymer concurrently withsaid contacting step, thereby forming a fibrin clot.

The subject invention also provides methods for preparing suchcomposition and compositions and kits comprising such fibrin monomer.

Another aspect of the subject invention relates to a method forutilizing a fibrin sealant which comprises:

(a) contacting a desired site with a composition comprisingnoncrosslinked fibrin; and

(b) converting said noncrosslinked fibrin to crosslinked:fibrinconcurrently with said contacting step, thereby forming a fibrin clot.

The subject invention also provides methods for preparing such acomposition and compositions and kits comprising such noncrosslinkedfibrin.

Definitions

For the purpose of the subject invention, the following definitions areutilized:

Fibrin--Fibrin means any form of fibrin. Nonlimiting examples of fibrininclude fibrin I, fibrin II and des BB fibrin. The fibrin can be inmonomeric form or polymeric form, wherein the polymeric form is eithernoncrosslinked or crosslinked.

Fibrin Monomer--Fibrin monomer includes any form of fibrin, e.g., fibrinI, fibrin II or des BB fibrin, wherein the fibrin is in monomeric formor oligomeric form that can be solubilized in the composition comprisingfibrin monomer and wherein the fibrin monomer can be converted to fibrinpolymer.

Fibrin Polymer--Fibrin polymer includes any form of fibrin, e.g., fibrinI, fibrin II or des BB fibrin, wherein said fibrin is in poly-mericform, either noncrosslinked or crosslinked.

Noncrosslinked Fibrin--Noncrosslinked fibrin includes any form offibrin, e.g., fibrin I, fibrin II or des BB fibrin, wherein said fibrinis noncrosslinked and can be converted to crosslinked fibrin. Thenoncrosslinked fibrin can be fibrin monomer or noncrosslinked fibrinpolymer.

Crosslinked Fibrin--Crosslinked fibrin includes any form of fibrin,e.g., fibrin I, fibrin II or des BB, wherein the fibrin is a fibrinpolymer that is crosslinked.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows activation of the support and immobilization ofthrombin-like enzyme on the support by coupling of the enzyme via --NH₂,--SH and --OH groups on the protein.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to a method for utilizing a fibrin sealantwhich comprises:

(a) contacting a desired site with a composition comprising fibrinmonomer; and

(b) converting said fibrin monomer to a fibrin polymer concurrently withsaid contacting step, thereby forming a fibrin clot.

The subject invention also provides methods for preparing suchcomposition and compositions and kits comprising such fibrin monomer.

Another aspect of the subject invention relates to a method forutilizing a fibrin sealant which comprises:

(a) contacting a desired site with a composition comprisingnoncrosslinked fibrin; and

(b) converting said noncrosslinked fibrin to crosslinked fibrinconcurrently with said contacting step, thereby forming a fibrin clot.

The subject invention also provides methods for preparing suchcomposition and compositions and kits comprising such noncrosslinkedfibrin.

The Composition Comprising Fibrin Monomer

The fibrin composition of the subject invention comprising fibrinmonomer is a composition that contains any form of fibrin monomer thatcan be converted to fibrin polymer. Nonlimiting examples of fibrinmonomer include fibrin I monomer, fibrin II monomer or des BB fibrinmonomer, with fibrin I monomer being preferred. Of course, mixtures ofthe fibrin monomer can be present. Also, for the purpose of the subjectinvention, fibrin polymer includes any polymer resulting from thepolymerization of fibrin monomer. Thus, for example, the conversion offibrin I monomer to fibrin polymer can result in fibrin I polymer,crosslinked or noncrosslinked, and/or fibrin II polymer, crosslinked ornoncrosslinked, depending on how the conversion step is carried out.

Fibrin I monomer is preferred because it can, in contrast to fibrinogen,readily be converted to fibrin polymer without the use of thrombin orfactor XIII. In fact, the fibrin I monomer can spontaneously form fibrinI polymer, which can act as the fibrin clot, regardless of whether thefibrin I polymer is crosslinked or noncrosslinked or further convertedto fibrin II polymer. Thus, since the formation of the fibrin I polymerfrom fibrin I monomer is spontaneous, the fibrin I polymer can be formedwithout thrombin and factor XIII, thereby avoiding the problemsassociated with bovine thrombin. (It should be noted that if fibrin Imonomer is utilized such that the fibrin I monomer comes into contactwith patient's blood, for example, on a wound, the patient's thrombinand factor XIII may convert the fibrin I polymer to crosslinked fibrinII polymer.)

The Composition Comprising Noncrosslinked Fibrin

The composition comprising noncrosslinked fibrin is a composition thatcontains any form of noncrosslinked fibrin. Nonlimiting examples ofnoncrosslinked fibrin are noncrosslinked fibrin I, noncrosslinked fibrinII and des BB fibrin, with noncrosslinked fibrin I being preferred. Ofcourse, mixtures of noncrosslinked fibrin can be present. Also, for thepurpose of the subject invention "crosslinked fibrin" includes any formof fibrin resulting from the conversion of noncrosslinked fibrin tocrosslinked fibrin. Thus, the crosslinked fibrin, for example, resultingfrom the conversion of noncrosslinked fibrin I to crosslinked fibrin,can be crosslinked fibrin I and/or crosslinked fibrin II, depending onhow the conversion step is carried out.

Noncrosslinked fibrin I is preferred because it can more readily, ascompared to fibrinogen, be converted to crosslinked fibrin. In fact, itis believed that fibrin I can form crosslinked fibrin I, which can actas the fibrin sealant. Thus, the formation of the crosslinked fibrin Ifrom noncrosslinked fibrin I can be carried out without thrombin,thereby avoiding the problems associated with bovine thrombin, albeitactivated factor XIII may be required. (It should be noted that ifnoncrosslinked fibrin I is utilized such that the noncrosslinked fibrinI comes into contact with patient's blood, for example, on a wound, thepatient's thrombin and factor XIII may convert the fibrin I tocrosslinked fibrin II.)

Also, the noncrosslinked fibrin can be a polymer, oligomer or monomer,albeit an oligomer or monomer is preferred, i.e., fibrin monomer. Thisis due to the fact that noncrosslinked fibrin in polymeric form isgenerally a gel and, therefore, is very difficult to deliver to thedesired site and provides for less intimate contact with cells at thedesired site. In contrast, a resulting noncrosslinked fibrin inoligomeric or monomeric form is soluble and, therefore, can more readilybe delivered to the desired site and have more intimate contact with thecells. Of course, the composition can contain a mixture of such forms ofnoncrosslinked fibrin.

The Source of the Composition Comprising Fibrin Monomer orNoncrosslinked Fibrin

The source of the composition comprising fibrin monomer ornoncrosslinked fibrin can be any source known or to be developed so longas the fibrin monomer can be converted to fibrin polymer or thenoncrosslinked fibrin can be converted to crosslinked fibrin.Nonlimiting sources of compositions comprising fibrin monomer ornoncrosslinked fibrin are blood, preferably mammalian blood and evenmore preferably human blood, cell cultures that secrete fibrinogen andrecombinant fibrinogen, with blood being preferred. Blood can be anyform of blood including, for example, whole blood or prepared fibrinogenpreparations. Also, blood can be utilized to prepare an autologousfibrin sealant.

It has been observed that a composition comprising noncrosslinked fibrinI, either as fibrin I monomer or fibrin I polymer, prepared from wholeblood as described below can be converted to crosslinked fibrin IIwithout the addition of thrombin, factor XIII and other necessarysubstances for blood coagulation| It is believed that this is due to thefact that the composition comprising noncrosslinked fibrin I preparedfrom whole blood retains sufficient quantities of prothrombin, factorXIII and such other necessary substances from the plasma such that thenoncrosslinked fibrin I can be converted to crosslinked fibrin IIwithout the addition of exogeneous thrombin and factor XIII. Thisendogenous prothrombin and factor XIII can be utilized in the fibrinsealant of the subject invention as components of the compositioncomprising fibrin monomer or noncrosslinked fibrin.

However, it should be noted that sufficient quantities of thisendogenous thrombin and factor XIII are not retained so as to convertfibrinogen to crosslinked fibrin II at a reaction rate that is suitablefor a fibrin sealant. It is believed that more thrombin is required toconvert fibrinogen to crosslinked fibrin II than to convertnoncrosslinked fibrin I to crosslinked fibrin II at an equivalentreaction rate.

Each one of such three sources contains fibrinogen, which can beconverted to the fibrin monomer or noncrosslinked fibrin. In addition tosuch conversion step, the resultant composition that contains the fibrinmonomer or noncrosslinked fibrin must be in a concentrated form. It ispreferred that the concentration of the fibrin monomer be no less thanabout 10 mg/ml, more preferably from about 20 mg/ml to about 200 mg/ml,even more preferably from about 20 mg/ml to about 100 mg/ml and mostpreferably from about 25 mg/ml to about 50 mg/ml.

In addition, it is preferred that the fibrin monomer or noncrosslinkedfibrin be nondynamic. For the purpose of the present invention anondynamic composition comprising noncrosslinked fibrin means that thenoncrosslinked fibrin in such composition does not crosslink for atleast about 1.5 minutes, preferably for at least about 3 minutes andmore preferably for at least about 30 minutes after preparation of suchcomposition. For the purpose of the present invention a nondynamiccomposition comprising fibrin monomer means that the fibrin monomer insuch composition does not polymerize for at least about 1.5 minutes,preferably for at least about 3 minutes, more preferably for at leastabout 30 minutes and even more preferably at least about 2 hours afterthe preparation of the composition. In fact, it should be noted that thecomposition comprising fibrin monomer can be nondynamic for at leastseveral days, i.e., about 72 hours, after its preparation|

Preparation and Concentration of a Composition Comprising Fibrin Monomeror Noncrosslinked Fibrin from Blood

The composition comprising fibrin monomer or noncrosslinked fibrin canbe prepared from blood. The method can result in a hydrogen bondedfibrin polymer, which is a form of noncrosslinked fibrin. Such polymercan then be utilized as a component of the fibrin sealant or beconverted to fibrin monomer by a process referred to as solubilization,all as described hereinbelow. Also, it is preferred that the compositioncomprising fibrin monomer or noncrosslinked fibrin be prepared in asterile environment.

Compositions comprising fibrin monomer or noncrosslinked fibrin can beprepared from whole blood by withdrawing blood from a donor andpreferably in the presence of an anticoagulant. Any anticoagulant can beutilized. Nonlimiting examples of anticoagulants are heparin, EDTA,hirudin, citrate or any other agent that can, directly or indirectly,prevent the formation of thrombin, with citrate being preferred.

The plasma, which contains the fibrinogen, is then separated from thewhole blood. Any separation technique can be utilized, for example,sedimentation, centrifugation or filtration. Centrifugation can becarried out at about 3,000 g. for about 10 minutes. However, if it isdesired to obtain plasma rich in platelets, centrifugation can becarried out at lower g force, e.g., 500 g for about 20 minutes. Thesupernatant, which contains the plasma, can be removed by standardtechniques.

Filtration can be carried out by passing the whole blood through asuitable filter that separates blood cells from plasma. It is preferredthat the filter be a microporous membrane exhibiting good proteintransmission.

The resultant plasma is then treated to convert the fibrinogen to fibrinmonomer or noncrosslinked fibrin. This conversion can be carried out byany technique that is known or to be developed.

A preferred technique to produce fibrin monomer or noncrosslinked fibrinis by means of a thrombin-like enzyme, which includes thrombin. Athrombin-like enzyme is any enzyme that can catalyze the formation offibrin from fibrinogen. A common source of thrombin-like enzymes aresnake venoms. Preferably, the thrombin-like enzyme is purified from thesnake venom. Depending on the choice of thrombin-like enzyme, suchthrombin-like enzyme can release fibrinopeptide A--which forms fibrinI--fibrinopeptide B--which forms des BB fibrin--or both fibrinopeptide Aand B--which forms fibrin II. It should be noted that thosethrombin-like enzymes that release fibrinopeptide A and B may do so atdifferent rates. Thus, the resultant composition could be, for example,a mixture of fibrin II and fibrin I or a mixture of fibrin II and des BBfibrin.

TABLE I is a nonlimiting list of the sources of the snake venoms thatcan be utilized in the subject invention, the name of the thrombin-likeenzyme and which fibrinopeptide(s) is released by treatment with theenzyme.

                  TABLE 1    ______________________________________                              FIBRINOPEPTIDE    SOURCE         NAME       RELEASED    ______________________________________    Agkistrodon acutus                   Acutin     A    A. contortrix  Venzyme    B, (A)*    contortrix    A. halys Pallas           B, (A)*    A. (Calloselasma)                   Ancrod, Arvin                              A    rhodostoma    Bothrops asper (B.                   Asperase   A    atrox)    B. Atrox       Batroxobin,                              A                   Reptilase    Reagent    B. insularis              A, B    B. jararaca    Botropase  A    B. Moojeni (B. Atrox)                   Batroxobin,                              A                   Defibrase    Lachesis muta muta        A, B    Crotalus adamanteus                   Crotalase  A    C. durissus terrificus    A    Trimeresurus   Flavoxobin A    flavoviridis    T. gramineus              A    Bitis gabonica Gabonase   A, B    ______________________________________     *() means low activity.

For a review of thrombin-like enzymes from snake venoms, see H. Pirkleand K. Stocker, Thrombosis and Haemostasis, 65(4):444-450 (1991).

The preferred thrombin-like enzymes are Batroxobin, especially from B.Moojeni, B. Maranhao and B. atrox and Ancrod, especially from A.rhodostoma.

The fibrin monomer or noncrosslinked fibrin can be prepared bycontacting the plasma with the thrombin-like enzyme, thereby permittingthe fibrinogen in the plasma to be converted to a fibrin monomer.However, the resultant fibrin monomer spontaneously polymerizes to forma hydrogen bonded polymer in the form of a gel, which separates from theremaining serum, which is a solution. The gel is a form of thecomposition comprising noncrosslinked fibrin.

This noncrosslinked fibrin gel can be harvested by, for example,centrifugation (3,000 g. for 10 minutes), direct manual separation ofthe noncrosslinked fibrin from the serum, filtration, directly or underpressure, followed by the removal of the separated serum. (A 1-100micron pore size filter can be utilized, for example, a sinteredpolypropylene 20 micron pore size filter from Porex, Inc., a teflon20-70 micron pore size filter from Fluorotechniques, Inc. or a nylon 6622-46 micron pore size filter from Costar; Inc.).

This harvestation separates the noncrosslinked fibrin gel from serum,which is a solution, and, thereby, concentrates the noncrosslinkedfibrin gel vis-a-vis the plasma. It should be noted that thenoncrosslinked fibrin gel retains at least some prothrombin, factorXIII, and such other necessary substances from the plasma such that thenoncrosslinked fibrin I can be converted to crosslinked fibrin IIwithout the addition of exogenous thrombin or factor XIII. Thisendogenous prothrombin and factor XIII can be utilized in the fibrinsealant of the subject invention as components of the compositioncomprising fibrin monomer or noncrosslinked fibrin.

The force of centrifugation or pressure of filtration duringharvettation will determine how much of the serum is removed from thenoncrosslinked fibrin gel, with the higher such force or pressure, themore concentrated the resulting noncrosslinked fibrin gel. However, itis preferred that such force or pressure not be so great that theprothrombin and factor XIII are removed from the noncrosslinked fibringel.

The noncrosslinked fibrin gel is now ready for use as a component of thefibrin sealant as a form of the composition comprising noncrosslinkedfibrin. It has been observed that when such composition is prepared fromwhole blood, from about 60% to about 90% of the original fibrinogen ispresent in the composition, but, of course, in the form of anoncrosslinked fibrin.

The composition comprising the noncrosslinked fibrin can be utilizedimmediately after it is prepared. In fact, it is particularly preferredto utilize such composition immediately after its preparation when thecomposition is autologous. If the composition is not utilizedimmediately after its preparation, the composition can be stored.Storage of the composition requires that the composition be preservedby, for example, freezing or lyophilizing the composition or holding thecomposition at 4° C. The composition in frozen or lyophilized form willbe stable for a period of months. When the composition is held at 4° C.,it is believed that the composition is stable for at least a period ofdays.

If the composition is frozen, the composition must be thawed prior tothe time of use.

This technique that results in the formation of the noncrosslinkedfibrin gel converts the fibrinogen to the noncrosslinked fibrin gel andconcentrates such gel in essentially one step.

Alternatively, and less preferred, one can concentrate fibrinogen byconventional techniques, e.g., cryoprecipitation and precipitation usingvarious reagents, e.g., polyethylene glycol, ether, ethanol, ammoniumsulfate or glycine. The concentrated fibrinogen can then be converted tothe noncrosslinked fibrin gel by the techniques described above or,preferrably, since the fibrinogen is already concentrated, then thefibrinogen can be converted to a composition comprising fibrin monomerwithout the need to first form the noncrosslinked fibrin gel. This canbe carried out by contacting the concentrated fibrinogen with achaotropic agent to obtain a fibrinogen solution.

The chaotropic agent is necessary to prevent the fibrin monomer, whichis formed upon contact of the fibrinogen with the thrombin-like enzyme,from spontaneously polymerizing. The chaotropic agent is mixed with suchfibrinogen composition and then agitated for about 1 to 2 minutes toform the fibrinogen solution. The fibrinogen can then be converted to afibrin monomer by, for example, a thrombin-like enzyme, as describedabove, or by a thrombin-like enzyme immobilized on a support, asdescribed below.

Suitable chaotropic agents include urea, sodium bromide, guanidinehydrochloride, KCNS, potassium iodide and potassium bromide. Thepreferred concentration of the chaotropic agent is from about 0.2 toabout 6.0 molar and most preferably from about 0.3 to about 2.0 molar.It is preferred to utilize the least amount of chaotropic agent possiblethat still prevents the fibrin monomer from spontaneously polymerizing.

It should be noted that it is preferred that a source of calcium ionsnot be added to the chaotropic agent until it is desired to convert thefibrin monomer to fibrin polymer, as described below. This ensures thatthe fibrin monomer will not crosslink due to activation of anyendogenous blood coagulation factors.

Immobilization of Thrombin-like Enzyme on a Support

In a preferred embodiment, the thrombin-like enzyme is immobilized on asupport. Such embodiment is preferred because one can readily separatethe immobilized enzyme from the plasma, thereby preventing thecomposition comprising noncrosslinked fibrin from being contaminated bythe enzyme.

Any support to which the thrombin-like enzyme can be attached can beutilized in the subject invention. Nonlimiting examples of suitablesupports are cellulose, polydextrans, agarose, polystyrenes, silica,polyacrylic acids, polyacrylamides, polyvinylalcohols, glass beads,polytetrafluorethylene, polycarbonate, collagen, celulose derivatives,teflon and their composites, with silica polystyrene and agarose beingpreferred and agarose being most preferred.

In another preferred embodiment the thrombinlike enzyme is attached to asupport that is a filter or on one side of the filter and attached toanother support, e.g., a bead. Otherwise, the thrombin-like enzyme willpass through the filter. Thus, the pore size of the filter should besuch that the immobilized thrombin-like enzyme cannot pass through thefilter, but the noncrosslinked fibrin can pass through the filter. Thenoncrosslinked fibrin is prepared by contacting the plasma with thethrombin-like enzyme on one side of the filter to form a fibrin monomer,which, along with the rest of the plasma, passes through the filter.Thus, the resulting composition comprising noncrosslinked fibrin isnecessarily separated from the thrombin-like enzyme. The fibrin monomer,after passage through the filter, spontaneously polymerizes to form anoncrosslinked fibrin polymer.

In order to immobilize the thrombin-like enzyme to a support, thesupport must be activated. This can be carried out by any suitabletechnique. For example, various activation chemistries available forderivatizing supports are: diazonium groups, isocyanate groups, acidchloride groups, acid anhydride groups, sulphonyl chloride groups,dinitro fluorophenyl groups, isothiocyanate groups, hydroxyl groups,amino groups, n-hydroxysuccinimide groups, triazine groups, hydrazinogroups, carbodiimide groups, silane groups and cyanogen bromide. See (a)Pentapharm Patent DT 2440 254 A1; (b) P.D.G. Dean, W. S. Johnson and F.A. Middle (Editors) (1991) IRL Press Oxford--Affinity Chromatography--Apractical approach-chapter 2--Activation Procedures, the disclosures ofwhich are incorporated herein by reference.

The preferred activation chemistry is by means of a hydrazide group. Theuse of a hydrazide activated support results in a maximal percentage (atleast about 30% to about 50% as measured by the S2238 assay--SeeAxelsson G. et al., Thromb. Haemost., 36:517(1976)) of the thrombin-likeenzyme retaining its activity with essentially no enzyme leaching. Also,low pH values, e.g., pH 4-6, can be utilized for enzyme coupling toprevent enzyme degradation.

Generally, the support is activated by a highly reactive compound, whichsubsequently reacts with a functional group of a ligand, e.g., --OH,--NH₂, --SH, --COOH, --CHO to form a covalent linkage. The preferredactivation chemistries for use in the subject invention are:

(a) by means of a triazine group and preferably triazine halogenidegroups;

(b) by means of a tresyl chloride group;

(c) by means of a carbonyldiimidazole group;

(d) by means of a cyanogen bromide group; and

(e) by means of a hydrazide or amino group.

In (a)-(d), the protein is coupled via reaction with --NH₂, --SH, or--OH groups, whereas in (e), the protein is coupled via an oxidizedcarbohydrate moiety, i.e., --CHO. The use of these chemistries resultsin a maximal percentage (at least about 30% to about 50% as measured bythe S2238 assay) of the thrombin-like enzyme retaining its activity withessentially no enzyme leaching.

For triazine activation two different methods were used. The firstmethod involved linking the triazine ring directly to surface OH groups.This is similar to the CNBr activation method employed, where surfacediol groups were reacted with CNBr. For triazine activation, OH groupsof agarose were reacted with triazine (cyanuric chloride).

Generally, the support is activated by a highly reactive compound, whichsubsequently reacts with a functional group of the ligand, e.g., --OH,--NH₂, --SH, --CHO, to form a covalent linkage. Remaining active groups,which have no thrombin-like enzyme attached, can be, but it is notessential, blocked with non-reactive compounds such as ethano-lamine,acetic anhydride or glycine.

The preferred activation chemistries for use in the subject inventionare:

(a) Cyanogen bromide activation followed by direct coupling of enzymevia --NH₂ groups on the protein.

(b) Activation of the support with monochlorotriazine followed bycoupling of an enzyme via --NH₂, --OH or --SH groups.

(c) Activation of the support with dichlorotriazine followed by couplingof the enzyme via --NH₂, --OH or --SH groups.

(d) Tresyl chloride activation of the support followed by coupling ofthe enzyme via --NH₂, --OH or --SH.

(e) Activation of the support with adipic acid hydrazide or hydrazinefollowed by coupling of oxidised enzyme via --CHO groups.

(f) Activation of the support with an amino ligand followed by couplingof oxidised enzyme via --CHO groups.

All the above preferred methodologies employ agarose as the support,however, it is possible to use silica. When using this support, thepreferred activation chemistries are:

(a) Gamma--glycidoxypropyltrimethoxysilane activation with directcoupling of the thrombin-like enzyme via --NH₂ groups on the protein.

(b) Cyanogen bromide activation followed by direct coupling of enzymevia --NH₂ groups on the protein.

(c) Gamma--glycidoxytrimethoxysilane activation followed by opening ofthe epoxide ring to form a diol group, which can be subsequentlyactivated with cyanogen bromide. Direct coupling of the enzyme can beachieved via --NH₂ groups on the protein.

(d) Gamma--glycidoxypropyltrimethoxysilane activation followed bypreparation of amino-silica by treatment with ammonia solution.

The amino-silica can be subsequently activated with cyanuric chloride(triazine) and the enzyme coupled via --NH₂, --OH or --SH groups.

Coupling of Enzyme to Activated Support

Coupling of the enzyme to the activated support must be buffered at acertain pH to obtain optimal enzyme binding. Generally, with standardactivation techniques such as gamma-glycidoxy-propyltri-methoxysilanecoupling of enzyme to activated support and cyanogen bromide coupling ofany protein to active groups requires buffering at a pH 1-2 units higherthan the pKa of the primary and secondary amines of the enzyme. However,the use of cyanuric chloride as the activator enables the use of muchlower pH buffers (optimal coupling pH is 4-6). Another method ofcoupling glycoproteins such as batroxobin to an inert support is viatheir carbohydrate moieties. This involves first the oxidation of thesugar group to --CHO groups followed by direct coupling at acid pH to anamino group such as hydrazide. A wide range of coupling buffers can beused. See, for example, Table 2.

                  TABLE 2    ______________________________________    EXAMPLES OF COUPLING BUFFERS USED IN ENZYME    IMMOBILIZATION TO SILICA AND AGAROSE SUPPORTS           ACTIVATION    SUPPORT           METHOD        COUPLING BUFFER    ______________________________________    Silica Gamma-        0.1M Sodium bicarbonate pH 8-9           glycidoxypropyl-                         10 mM HEPES pH 7.0           trimethoxysilane    Silica γ-glycidoxypropyl-                         0.1M Sodium bicarbonate pH 8-9           trimethoxysilane +                         10 mM HEPES pH 7.0           cyanogen bromide    Silica Cyanogen Bromide                         Water pH 7.0                         0.1M Sodium bicarbonate pH 7-9                         10 mM HEPES pH 7.0    Agarose           Monochlorotriazine                         50 mM Sodium Acetate/1MNaCl pH                         4.0    Agarose           Dichlorotriazine                         0.1M Potassium                         phosphate/1MNaCl pH 8.0-9.0    Agarose           Tresyl chloride                         50 mM Potassium                         phosphate/0.5MNaCl pH 7.7    Agarose           Hydrazide     50 mM Sodium Acetate pH 5.5                         10 mM NaBH.sub.4    Agarose           Amine         50 mM Sodium Acetate pH 5.5                         10 mM NaBH.sub.4    ______________________________________

Blocking and Deactivating Remaining Active Groups

After activation the support will possess more active sites thanrequired for enzyme coupling. These sites, if not deactivated, maycovalently bind contaminating proteins, which might affect thebiological function of the immobilized enzyme.

Excess groups can be deactivated by the covalent coupling of small,noninterfering amines such as ethanolamine.

If hydrazide or amino activated supports are employed, blocking ofresidual reactive groups following enzyme coupling can be acheived byuse of acetic anhydride.

Depending on the method of immobilization and the support, enzymeinactivation occurs during the immobilization process. Employing asilica support with the most desirable activation chemistry (e.g.,cyanogen bromide), up to 80-90% of enzyme activity is lost. However, theuse of agarose as the support with cyanuric chloride activation resultsin less loss of of enzyme activity.

Characterization

In order to characterize the efficacy of the immobilized enzyme on thesupport, two methods can be utilized to assess the amount of activeenzyme immobilized on the support; the Clot Time Assay--Clauss A., ActaHaematol., 11:237 (1957) and the S2238 Assay--See Axelsson G. et al.,Thromb. Haemost., 36:517 (1976).

To assess the leaching of the enzyme from the support, the followingassay can be utilized.

Leaching can be assayed by radiolabelling the thrombin-like enzyme,e.g., I¹²⁵ batroxobin. However, prior to carrying out the leachingassay, it is necessary to remove any unbound radiolabel. This can beachieved by sequentially washing the support with: 50 ml 50 mM sodiumacetate pH 5.5, 100 ml 50 mM glycine/1M sodium chloride pH 3.0, 100 ml50 mm sodium carbonate/1M sodium chloride pH 10.0, 100 ml 50 mM sodiumphosphate/lM sodium chloride pH 7.0, 100 ml water and 100 ml 50 mMsodium phosphate/1M sodium chloride pH 7.0. After such a washingprocedure, there was no detectable radiolabel in the washings, with ≧50%of the initial radiolabelled enzyme coupled to the support.

Formation of Noncrosslinked Fibrin

(a) Amount of Enzyme Required

The amount of enzyme required for the treatment of 30-70 ml plasma(obtained from 60-150 ml whole blood) is 30-200 units after about 10-15minutes mixing.

If agarose is employed as the support matrix, 30-200 U of batroxobinresults in the formation of noncrosslinked fibrin in about 7-20 minutes.This system employs hydrazide activation and 0.25 g-1.0 g of dryagarose. In this case no blocking of remaining active groups isrequired.

(b) Reaction of Immobilized Enzyme with Plasma Fibrinogen

Generally, the reaction of the immobilized enzyme with fibrinogen inplasma is performed as follows: approximately 30-70 ml of plasma isadded to a known quantity of a dried support, which contains theimmobilized thrombin-like enzyme. The suspension is mixed gently(rotation on a spiral mixer or hand mixing) for approximately 7-20minutes. During this time fibrinogen in the plasma is cleaved by theimmobilized enzyme to release fibrinopeptide A and/or fibrinopeptide Bresulting in the formation of a hydrogen bonded fibrin I polymer,hydrogen bonded des BB fibrin polymer or hydrogen bonded fibrin IIpolymer wherein each polymer is associated with the immobilized enzyme.

As described above, the hydrogen bonded fibrin polymer is in the form ofa gel and can be harvested by, for example, centrifugation (3,000 g. for10 minutes) or filtration (through a 1-50 micron membrane filter). Suchharvestation separates the hydrogen bonded fibrin polymer from serumand, thereby, concentrates the polymer.

The Prevention of Formation of Crosslinked Fibrin

It should be noted that if a thrombin-like enzyme is utilized in plasmathat results in the activation of factor XIII, then it is preferred thatthe plasma composition be modified at the time the thrombin-like enzymeis utilized so as to prevent the noncrosslinked fibrin, e.g.,noncrosslinked fibrin I or II, from forming crosslinked fibrin, e.g.,crosslinked fibrin I or II. Of course, it may not be necessary to modifythe plasma composition if such composition is utilized as a fibrinsealant immediately after the fibrinogen has been converted tononcrosslinked fibrin.

The plasma composition can be modified to prevent the crosslinking ofthe noncrosslinked fibrin by any technique that is known or to bedeveloped. This can be carried out by blocking the endogenous thrombinthat can activate the factor XIII, e.g., hirudin or thrombin inhibitors,or blocking the action of activated factor XIII, e.g., by means of heavymetals (Hg), thiomerosal or inhibitory antibodies. Crosslinking offibrin I or II requires the presence of calcium ions. Thus, if thecalcium is removed from the plasma composition, crosslinking of thefibrin I or II can be inhibited. See Carr et al., J. Biochem., 239:513(1986); Kaminski et al., J. Biol. Chem. 258:10530 (1983) and Kanaide etal. 13:229 (1982). Calcium chelators can be added to the composition toprevent the crosslinking of the fibrin I or II. Such chelators bind tothe calcium, thereby preventing the crosslinking. Any calcium chelatorcan be utilized. Nonlimiting examples of calcium chelators includecitric acid, saccharic acid, ethylenediaminetetraacetic acid (EDTA),nitri-lotriacetic acid (NTA), hydroxyethylene-diaminetriacetic acid(HEEDTA), ethylenediaminedi- o-hydroxyphenylacetic acid! (EDDHA),ethylene-glycolbis (2-aminoethylether) tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA),1,2-diaminocyclohexanetetraacetic acid (DCTA),N,N-bishydroxyethylglycine,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) andN-hydroxyethyliminodiacetic acid (HIMDA) and salts thereof, with saltsof citric acid being preferred.

Preparation and Concentration of Fibrin Monomer or Noncrosslinked Fibrinfrom Cell Cultures that Secrete Fibrinogen

Any cell culture that can secrete fibrinogen can be utilized in thesubject invention. The fibrinogen in the cell culture can be convertedto fibrin monomer or noncrosslinked fibrin by the same techniques asthose described above with respect to plasma. However, prior to suchconversion, it is preferred that the cellular debris be removed.

The process can be carried out as follows: HEPG2 cells are grown andmaintained as described by standard texts on mammalian cell culture.Also, see Liu et al., Cytotechnology, 5:129-139 (1991). The cells areseeded into flasks at a split ratio between 1:4 to 1:8 in MinimalEssential Medium containing 10% calf serum and buffered with 5% CO₂.

After 24-36 h growth at 37° C., the medium is removed and replaced withserum free medium containing a suitable protease inhibitor and 2 IU/mlheparin. Culture is continued for further 24 h periods with threeconsecutive changes of serum free media.

The conditioned media is centrifuged at 3,000 g for 10 minutes to removeany cell debris and the clarified supernatant, which containsfibrinogen, is then gently mixed with a thrombin-like enzyme for 4-5hours. Preferably, the thrombin-like enzyme is immobilized. A ratio offrom about 1.0 ml to about 50 ml., settled volume agarose-thrombin-likeenzyme per 500 ml media is suitable. As described above, supports otherthan agarose can be utilized. The resulting fibrin monomer spontaneouslypolymerizes and is enmeshed around the immobile support.

The supernatant, which now contains no fibrinogen, is decanted from thesupport/fibrin gel, which is then washed successfully with four changesof NaCl-0.15 M. at a ratio of from about 10 ml. to about 100 ml. per 1.0ml original settled volume of support. The washed gel is thensemi-dehydrated using a sintered glass funnel under vacuum.

The use of cell cultures that can secrete fibrinogen are particularlypreferred when a composition comprising a fibrin monomer is utilized.This is because it is believed that such composition can be utilized toform a fibrin polymer that is useful as a fibrin sealant, regardless ofwhether the fibrin polymer ultimately crosslinks. Thus, since such cellculture does not contain factor XIII, which is essential forcrosslinking, no exogenous factor XIII need be added to make aneffective fibrin sealant.

Preparation and Concentration of Fibrin Monomer or Noncrosslinked Fibrinfrom Recombinant Fibrinogen

The noncrosslinked fibrin composition can also be prepared fromrecombinant fibrinogen. Only recently fibrinogen has been made byrecombinant DNA techniques. See S. Roy et al., The Journal of BiologicalChemistry, 266:4758-4763 (1991), the contents of which are incorporatedherein by reference. Roy et al. appears to be the first group to expressall three chains of fibrinogen and teach that COS cells express,assemble and secrete the chains in a form that is capable of forming athrombin-induced clot. The resultant fibrinogen composition can then beutilized to produce the composition comprising fibrin monomer ornoncrosslinked fibrin by the same techniques as described hereinabovewith respect to cell cultures that secrete fibrinogen. However, it ispreferred that prior to the formation of the composition comprisingfibrin monomer or noncrosslinked fibrin, the cellular debris be removedby the same techniques as described above with respect to cell cultures.The cellular debris can be removed by centrifugation or filtration.

The use of recombinant fibrinogen is particularly preferred when acomposition comprising a fibrin monomer is utilized. This is because itis believed that such composition can be utilized to form a fibrinpolymer that is useful as a fibrin sealant, regardless of whether thefibrin polymer ultimately crosslinks. Since the recombinant fibrinogencell culture does not contain factor XIII, which is essential forcrosslinking, no exogenous factor XIII need be added to make aneffective fibrin sealant.

Solubilization of Noncrosslinked Fibrin

The conversion of fibrinogen to noncrosslinked fibrin by, for example, athrombin-like enzyme, results in the formation of the fibrin in the formof a fibrin hydrogen-bonded polymer. However, as discussed above, it ispreferred that the composition comprising noncrosslinked fibrin, and itis essential for the composition comprising fibrin monomer, be inoligomeric or monomeric form. This can be carried out by solubilizationof the composition comprising noncrosslinked fibrin.

Solubilization is particularly preferred when the noncrosslinked fibrinis formed by means of a thrombin-like enzyme that is immobilized on asupport. This is due to the fact that the use of a thrombin-like enzymeimmobilized on a support generally results in the noncrosslinked fibrinhydrogen-bonded polymer being associated with such immobilized enzyme.Thus, since it is preferred that the support not be contained in theresulting composition, solubilization permits one to also remove fromthe composition comprising noncrosslinked fibrin the support, along withthe immobilized enzyme.

Solubilization can be carried out by any technique that is known or tobe developed that results in a fibrin monomer. Solubilization can becarried out by contacting the composition comprising noncrosslinkedfibrin with a suitable acid buffer solution, preferably an acid bufferhaving a pH of less than about 5 and preferably from about 1 to about 5.Nonlimiting examples of suitable acid buffer solutions include aceticacid, succinic acid, glucuronic acid, cysteic acid, crotonic acid,itaconic acid, glutamic acid, formic acid, aspartic acid, adipic acidand salts thereof and with succinic acid, aspartic acid, adipic acid andsalts of acetic acid being preferred and most preferably sodium acetate.It has been observed that the preferred acid buffers functioned muchmore efficiently than that of other acid buffers that were tested.

The preferred concentration of the acid buffer is from about 0.02 M toabout 1 M and most preferably from about 0.1 M to about 0.3 M. Suchpreferred concentration renders the ionic strength of the compositionmore biologically compatible.

It is preferred to utilize the least volume of acid buffer possible thatstill solubilizes the noncrosslinked fibrin to form an aqueous solutioncomprising fibrin monomer. This results in an aqueous solutioncomprising fibrin monomer that is highly concentrated in fibrin monomer.Generally, from about 1 ml to about 4 ml of acid buffer per about 1 mlof composition comprising noncrosslinked fibrin is required.

The acid buffer is mixed with the noncrosslinked fibrin and thenagitated vigorously for about 1 to 2 minutes to ensure thatsolubilization is complete.

Solubilization can also be carried out at neutral pH by means of achaotropic agent. Suitable chaotropic agents include urea, sodiumbromide, guanidine hydrochloride, KCNS, potassium iodide and potassiumbromide. The preferred concentration of the chaotropic agent is fromabout 0.2 to about 6.0 molar and most preferably from about 3.5 to about5.0 molar.

As with the acid buffer, it is preferred to utilize the least amount ofchaotropic agent possible that still solubilizes the noncrosslinkedfibrin. Generally, from about 1.0 ml to about 1.5 ml of chaotropic agentper about 1 ml of composition comprising noncrosslinked fibrin isrequired.

The chaotropic agent is mixed with such composition and then agitatedvigorously for about 1 to 2 minutes to ensure that solubilization iscomplete.

Accordingly, solubilization results in a composition comprising a fibrinmonomer and, in particular, an aqueous solution comprising fibrinmonomer. It is preferred that the fibrin monomer concentration in theaqueous solution be no less than about 10 mg/ml, more preferably fromabout 20 mg/ml to about 200 mg/ml, even more preferably from about 20mg/ml to about 100 mg/ml and most preferably from about 25 mg/ml toabout 50 mg/ml.

If a thrombin-like enzyme immobilized on a support is utilized thatresults in such enzyme being present in the composition comprisingnoncrosslinked fibrin, after solubilization the immobilized enzyme canbe removed from the composition comprising the fibrin monomer. This canbe carried out by, for example, filtration through any suitable filterthat can separate the enzyme. Suitable filters include a sinteredpolypropylene 20 micron pore size filter from Porex, Inc., a teflon20-70 micron pore size filter from Fluorotechniques, Inc. or a nylon 6622-46 micron pore size filter from Costar, Inc.

An alternative method for ensuring that no thrombin-like enzyme, e.g.,batroxobin, is present in the composition comprising fibrin monomer isto use a soluble thrombin-like enzyme in the system and remove theenzyme following solubilization of the fibrin hydrogen-bonded polymer.The removal of the enzyme can be achieved by use of an affinity matrix,e.g., a ligand bound to an inert support that has a specific affinityfor the thrombin-like enzyme or an ion exchange or hydrophobicinteraction support or most effectively using the avidin-biotin system.The biotin-avidin interaction exhibits one of the strongest non-covalentbinding constants (K_(DIS=) 10⁻¹⁵ M) seen in nature. See E. A. Bayer andM. Wilchek, Methods of Biochemical Anaylsis, 26:1(1980).

In this process, biotin is covalently bound to, for example, batroxobinand the biotin-batroxobin conjugate (which is soluble) is directlyreacted with plasma, e.g., 10 BU plus 50 ml plasma reacted at 37° C. for10 minutes. The Fibrin I polymer produced is harvested by centrifugationor filtration and resolubilized in approximately 4 ml 0.2M sodiumacetate pH 4.0 containing 30 mM calcium chloride. To the Fibrin Isolution is added a molar excess of avidin coupled to an inert supportsuch as agarose. The agarose:avidin:biotin-batroxobin complex is thenseparated from the Fibrin I by centrifugation or filtration resulting ina composition comprising fibrin monomer that is substantially free ofbatroxobin, which can be repolymerised as described below to generate afibrin sealant.

Accordingly, in one embodiment of the present invention, the compositioncomprising fibrin monomer is substantially free of the thrombin-likeenzyme. By substantially free is meant either that all of thethrombin-like enzyme has been removed, or that any thrombin-like enzymeremaining in the composition is at levels insufficient to provide anyundesired pharmacological effect. Thus, compositions of this inventiondesired to be "substantially free" may contain thrombin-like enzyme inan amount between about zero and 10 percent of the original enzyme andpreferably between about zero and 2 percent of the thrombin-like enzymeused to prepare the fibrin monomer composition.

Although these embodiments describe compositions wherein thethrombin-like enzyme has been removed following the desired conversionto soluble fibrin, compositions retaining most or all of thethrombin-like enzyme are also believed useful and, as such, are a partof the present invention.

The composition comprising fibrin monomer is now ready for use as acomponent of the fibrin sealant. It has been observed that when suchcomposition is prepared from whole blood, from about 60% to about 90% ofthe original fibrinogen is present in the composition, but, of course,in the form of fibrin monomer. The composition comprising the fibrinmonomer can be utilized immediately after it is prepared. In fact, it isparticularly preferred to utilize such composition immediately after itspreparation when the composition is autologous. If the composition isnot utilized immediately after its preparation, the composition can bestored. Storage of the composition requires that the composition bepreserved by, for example, freezing or lyophilizing the composition orholding the composition at 4° C. It is believed that the composition infrozen or lyophilized form will be stable for a period of months. Whenthe composition is held at 4° C., it is believed that the composition isstable for at least a period of days.

If the composition is frozen, the composition must be thawed at the timeof use. If the composition is lyophilized, at time of use, it ispreferred that the composition be reconstituted by the addition of thesame acid buffer that was utilized in the solubilization step if thatacid was volatile, e.g., acetic acid, or if a choatropic agent wasutilized, by the addition of distilled water. AS in the solubilizationstep, in reconstitution the least amount of acid buffer solution ordistilled water should be utilized that still results in the fibrinmonomer being soluble. In fact, reconstituting a lyophilized compositioncomprising fibrin monomer can result in an aqueous solution comprisingfibrin monomer wherein such monomer concentration is up to 200 mg/ml.Prior to lyophilization, a bulking agent, e.g., mannitol or lactose, canbe added to the composition. Alternatively, the lyophilized compositioncan be utilized in lyophilized form. Such form is particularly preferredwhen it is desired to add adjuvants, e.g., antibiotics, to thecomposition.

The composition comprising fibrin monomer can be in virtually any form,for example, a solution, suspension, emulsion or solid, with a solutionbeing preferred. Thus, for example, such composition can be a liquid,gel, paste or salve. Also, of course, the composition can be in the formof a "granule," for example, lyophilized fibrin monomer, that can be,but need not be, treated to form a solution, emulsion or suspensionimmediately prior to use.

Any suitable solvent can be utilized to form the solution, but, ofcourse, it is preferred that the solvent be nontoxic. Nonlimitingexamples of solvents include water, ethyl alcohol, glycerol andpropylene glycol, with water being preferred.

An example of a suspension is that the composition comprising fibrinmonomer can be mixed with organic solvents, e.g., ethanol, to a finalethanol concentration in excess of 3.0 M and shaken. The fibrin monomerwill precipitate and can be recovered by centrifugation. The precipitateshould be washed with organic phase to remove the aqueous solution usedin the solubilization step. An ethanolic suspension of monomeric fibrincan then be applied directly to a bandage or other carrier or evenapplied directly to a wound site. The organic phase is allowed toevaporate. In the case of a bandage or other carrier the suspension canbe rehydrated by contacting with the site of application or some othermeans, and the fibrin allowed to polymerize.

Alternatively, an organic suspension of fibrin monomer prepared in ahighly volatile phase, e.g., diethyl ether, might be atomized anddelivered as a spray suspension to the desired site. It is preferablethat the volatile phase is non-flammable. It is possible that an organicsuspension of fibrin monomer could be delivered to the ear, nose,throat, or lungs by spray or breathing or delivered to a bleedingoesophageal or gastric lesion by injestion.

The uses of the Fibrin Sealant of the Subject Invention

The fibrin sealant of the subject invention is utilized by contactingthe desired site with the composition comprising fibrin monomer ornoncrosslinked fibrin and converting the fibrin monomer to fibrinpolymer or noncrosslinked fibrin to crosslinked fibrin concurrently withsaid contacting step, thereby forming the fibrin clot.

For the purpose of the subject invention "desired site" is that locationwhere one desires to form the fibrin clot. What or where the desiredsite is depends on the use of the fibrin sealant of the subjectinvention. Also, it should be noted that it is believed that the fibrinsealant can be utilized not only in humans but also in other mammals.Also, if the source of the fibrin sealant is blood, then it ispreferred, but not essential, that the blood be derived from the samespecies that the fibrin sealant will be utilized.

The fibrin sealant of the subject invention can be utilized for any usethat is known or to be developed for a fibrin sealant. The methods, kitsor fibrin sealant of the subject invention can be used for connectingtissues or organs, stopping bleeding, healing wounds, sealing a surgicalwound, use in vascular surgery include providing hemostasis for stitchhole bleeding of distal coronary artery anastomoses; left ventricularsuture lines; aortotomy and cannulation sites; diffuse epimyocardialbleeding seen in reoperations; and oozing from venous bleeding sites,e.g. at atrial, caval, or right ventricular levels. The subjectinvention is also useful for sealing of dacron artery grafts prior tografting, sealing tissues outside the body, producing fibrin rafts forcell growth, stopping bleeding from damaged spleens (thereby saving theorgan), livers, and other parenchymatous organs; sealing tracheal andbronchial anastomoses and air leaks or lacerations of the lung, sealingbronchial stumps, bronchial fistulas and esophageal fistulas; forsutureless seamless healing ("Zipper" technique), and embolization invascular radiology of intracerebral AVM's, liver AVM's, angiodysplasiaof colon, esophageal varices, "pumping" GI bleeders secondary to pepticulcers, etc. The subject invention is further useful for providinghemostasis in corneal transplants, nosebleeds, post tonsillectomies,teeth extractions and other applications. See G. F. Gestring and R.Lermer, Vascular Surgery, 294-304, September/October 1983. Also, thefibrin sealant of the subject invention is especially suited forindividuals with coagulation defects.

The dosage of the composition comprising fibrin monomer or compositioncomprising noncrosslinked fibrin depends on the particular use of thefibrin sealant, but the dosage should be an effective amount for thecomposition to perform its intended use. Generally, for a compositioncomprising fibrin monomer that is an aqueous solution, it is believedthat from about 3 ml to about 5 ml of such composition is sufficient tobe an effective fibrin sealant. However, depending on the use of thecomposition, the dosage can range from about 0.05 ml to about 40 mlAlso, if a composition comprising noncrosslinked fibrin in polymer formis utilized or the composition is in solid form, then the compositionshould contain that amount of fibrin that is in such aqueous solution.

The Administration of the Fibrin Sealant of the Subject Invention

For the purpose of the subject invention the conversion of the fibrinmonomer to fibrin polymer or noncrosslinked fibrin to crosslinked fibrin"concurrently" with said contacting step means that such conversion stepand such contacting step occur within a time period of each step so asto form the fibrin clot at the desired site. Thus, concurrently can meanthat after the contacting step, the fibrin monomer is converted tofibrin polymer or the noncrosslinked fibrin is converted to crosslinkedfibrin. This is carried out by contacting the composition comprisingfibrin monomer or noncrosslinked fibrin, after such composition has beenapplied to the desired site, with a composition that can convert thefibrin monomer to fibrin polymer or the noncrosslinked fibrin tocrosslinked fibrin. The conversion step should generally occur withinabout 0.5 minutes after the contacting step. Otherwise, the compositioncomprising the fibrin monomer or noncrosslinked fibrin, especially ifthe noncrosslinked fibrin is a fibrin monomer, may flow away from thedesired site.

Concurrently can also mean that the contacting step and converting steptake place simultaneously. This is carried out by contacting the desiredsite with the composition comprising the fibrin monomer ornoncrosslinked fibrin at the same time that such composition iscontacted with a composition that can convert the fibrin monomer tofibrin polymer or the noncrosslinked fibrin to crosslinked fibrin.

Finally, and preferably, concurrently can also mean that the conversionstep can commence prior to the contacting step, albeit not so far priorto the contacting step that all of the fibrin monomer has been convertedto fibrin polymer or all of the noncrosslinked fibrin has been convertedto crosslinked fibrin. Otherwise, all of the fibrin monomer will beconverted to fibrin polymer or all of the noncrosslinked fibrin will beconverted to crosslinked fibrin, prior to the contacting step, whichresults in a very poor fibrin sealant. This embodiment is carried out bymixing the composition comprising the fibrin monomer or noncrosslinkedfibrin with a composition that can convert the fibrin monomer to fibrinpolymer or the noncrosslinked fibrin to crosslinked fibrin, prior to thecontacting step. Since it takes about 30 seconds for the conversion stepto be complete, the conversion step should not begin more than about 30seconds and preferably not more than about 3 seconds prior to thecontacting step. This embodiment is preferred because it ensures thatthe maximum amount of the composition comprising the fibrin monomer ornoncrosslinked fibrin will remain at the desired site and yet also formsan excellent fibrin clot.

Conversion of the Composition Comprising Fibrin Monomer to FibrinPolymer or Noncrosslinked Fibrin to Crosslinked Fibrin

The conversion of the fibrin monomer to fibrin polymer or thenoncrosslinked fibrin to crosslinked fibrin can be carried out by anytechnique that is known or to be developed. However, how the conversionstep is carried out depends on the source of the composition comprisingfibrin monomer or noncrosslinked fibrin e.g., whole blood or recombinantfibrinogen, the form of the fibrin monomer or noncrosslinked fibrin,e.g., fibrin I or des BB fibrin, and, to a lesser extent, whether thedesired site will contain the patients blood or other body fluids at thetime of use of the fibrin sealant.

Also, if a separate composition, such as an alkaline buffer, is utilized(discussed below) for the conversion step, then the method of thesubject invention can be carried out with, for example, adouble-barrelled syringe. The double-barrelled syringe can be Y-shaped,thereby permitting the mixing of the composition comprising fibrinmonomer or noncrosslinked fibrin and the composition to be utilized inthe conversion step to mix immediately prior to the contacting step.Also, rather than a Y-shaped double-barrelled syringe a double-barrelledsyringe with two openings can be utilized. This permits the simultaneouscontacting of the desired site and conversion to fibrin polymer orcrosslinked fibrin to take place. Also, the compositions of thedouble-barrelled syringe can be sprayed onto the desired site. See H. B.Kram et al., The American Surgeon, 57:381 (1991).

Blood as the Source of the Composition Comprising Fibrin Monomer orNoncrosslinked Fibrin

If the source of the composition comprising fibrin monomer ornoncrosslinked fibrin is blood, then as discussed above, it is believedthat the composition will retain enough prothrombin, factor XIII andother necessary substances to convert such fibrin monomer ornoncrosslinked fibrin to crosslinked fibrin, e.g., activators ofprothrombin. If the noncrosslinked fibrin is a fibrin polymer, i.e., thenoncrosslinked fibrin has not been solubilized, the noncrosslinkedfibrin can be converted to crosslinked fibrin by the activation ofprothrombin and factor XIII of such composition to form crosslinkedfibrin. Such activation can be carried out by contacting the compositionwith a source of calcium ions. Nonlimiting sources of calcium ionsinclude calcium chloride, preferably at a concentration of 30 mM.Alternatively, and less preferred, the calcium ions can be supplied bythe blood at the desired site.

If the noncrosslinked fibrin has been solubilized, i.e., is a fibrinmonomer, how the fibrin monomer is converted to crosslinked fibrindepends on how the solubilization was carried out. If the noncrosslinkedfibrin was solubilized by an acid buffer, the crosslinked fibrin can beformed by raising the pH of the composition comprising fibrin monomersuch that the fibrin monomer can polymerize. This can be carried out bycontacting such composition with any suitable alkaline buffer.Nonlimiting examples of suitable alkaline buffers include HEPES, sodiumhydroxide, potassium hydroxide, calcium hydroxide, bicarbonate bufferssuch as sodium bicarbonate and potassium bicarbonate, tri-metal salts ofcitric acid, salts of acetic acid and salts of sulfuric acid. Preferredalkaline buffers include: 0.5-0.75M Sodium carbonate/bicarbonate pH10-10.5, 0.5-0.75M Sodium bicarbonate/NaOH pH 10.0, 1.5M Glycine/NaOH pH10.0, 0.5-1.0 M Bis hydroxeythylaminoethane sulphonic acid (BES) pH 7.5,1M Hydroxyethylpiperazine propane sulphonic acid (EPPS) pH 8.5, 0.5MTricine pH 8.5, 1M Morpholino propane sulphonic acid (MOPS) pH 8.0, 1MTrishydroxymethyl aminoethane sulphonic acid (TES) pH 8.0 and 0.5MCyclohexylaminoethane sulphonic acid (CHES) pH 10.0; with 0.5-0.75MSodium carbonate/bicarbonate pH 10-10.5, 0.5-1.0M Bishydroxeythylaminoethane sulphonic acid (BES) pH 7.5, 1MHydroxyethylpiperazine propane sulphonic acid (EPPS) pH 8.5 and 1MTrishydroxymethyl aminoethane sulphonic acid (TES) pH 8.0 being mostpreferred.

The amount of alkaline buffer that is utilized should be enough topolymerize the noncrosslinked fibrin. It is preferred that about 10parts to about one part of composition comprising fibrin monomer bemixed with about 1 part alkaline buffer. It is even ore preferred thatsuch ratio be about 9:1. It should be noted that the preferred ratiodepends on the choice of buffer and the desired "strength" of the fibrinpolymer. Of course, the desired strength of the fibrin polymer dependson the end-use of the fibrin sealant.

If the solubilization was carried out with a chaotropic agent, then thefibrin monomer can be converted to crosslinked fibrin by diluting thecomposition comprising fibrin monomer with, for example, distilledwater. The dilution should be carried out such that the minimal amountof diluent is utilized. Generally; the resulting concentration of thechaotropic agent after dilution should be from about 0.5 to about 0.1molar.

In addition to raising the pH or diluting the chaotropic agent of thecomposition comprising fibrin monomer, it is preferred that theprothrombin and factor XIII of such composition be activated to form thecrosslinked fibrin. Such activation can be carried out by the contactingthe composition with a source of calcium ions. The source of the calciumions can be part of the alkaline buffer or part of the acid buffer thatis utilized in the solubilization step. Nonlimiting sources of calciumions include calcium chloride, preferably at a concentration of 30 mM.Alternatively, and less preferred, the source of calcium ions can besupplied by the blood at the desired site.

It should be noted that if the alkaline buffer is acarbonate/bicarbonate buffer, then the source of calcium ions must beadded to the acid buffer during the solubilization step. This is due tothe fact that the calcium chloride is not soluble in thecarbonate/bicarbonate buffer. It is preferred that the concentration ofcalcium ions in the acid buffer solution be from about 5 millimolar toabout 150 millimolar and more preferably from about 5 mM to about 50 mM.

It is believed that the resulting fibrin clot will be crosslinked fibrinII, regardless of which form of noncrosslinked fibrin, i.e., fibrinmonomer or fibrin polymer, is present. However, if the form ofnoncrosslinked fibrin is des BB fibrin, then it is believed that inaddition a source of additional thrombin may be required to convert desBB fibrin to crosslinked fibrin. Such a source of thrombin can be, forexample, plasma from the patient wherein such plasma is added to thecomposition comprising noncrosslinked fibrin.

If the desired site contains blood and a composition comprising a fibrinmonomer is utilized, i.e., the noncrosslinked fibrin has beensolubilized, then this blood can be utilized as a diluent of thechaotropic agent or to raise the pH of the composition comprising fibrinmonomer. Thus, no diluent or alkaline buffer need be utilized. In thisembodiment, it is preferred that the source of calcium ions be containedin the acid buffer or chaotropic agent utilized in the solubilizationstep. Also, in this embodiment the composition comprising fibrin monomercan be placed on a solid support, e.g., bandage, suture, prosthesis, ordressing, that will be in contact with the desired site. Such support isthen placed in contact with the desired site until, for example, thefibrin clot forms.

However, it should be noted, that if the composition comprising fibrinmonomer does not retain enough prothrombin and factor XIII so as to formcrosslinked fibrin, such composition is still useful as a fibrin sealantbecause the polymerization of fibrin monomer per se is useful to form afibrin clot. Also, such composition can still be utilized to formcrosslinked fibrin by the addition of a source of calcium ions andactivated factor XIII (or precursors to activated factor XIII) and,optionally, thrombin. Such source of calcium ions, activated factor XIIIand thrombin can be added to the compositions comprising fibrin monomer.The activated factor XIII can be added to the composition comprisingfibrin monomer at a final concentration of from about 1.0 to about 20units factor XIII per ml of composition comprising noncrosslinkedfibrin. Alternatively, the factor XIII can be supplied by the blood orbody fluids at the desired site or by the addition of autologous plasmato the composition comprising fibrin monomer. Nonlimiting sources ofcalcium ions include calcium chloride, preferably at a concentration of30 mM. Alternatively, and less preferred, calcium ions can be suppliedby the blood or body fluids at the desired site. From about 4 units toabout 500 units of thrombin per ml. of composition comprising fibrinmonomer can be added or the thrombin can be provided by the desiredsite.

Cell Cultures that Secrete Fibrinogen or Recombinant Fibrinogen as theSource of the Composition Comprising Fibrin Monomer or NoncrosslinkedFibrin

If the source of the composition comprising noncrosslinked fibrin arecell cultures that secrete fibrinogen or recombinant fibrinogen, and thenoncrosslinked fibrin is a fibrin polymer, i.e., the noncrosslinkedfibrin has not been solubilized, then a source of calcium ions andactivated factor XIII (or precursors to activated factor XIII) must beutilized to form crosslinked fibrin. Factor XIII must be utilizedbecause these sources of noncrosslinked fibrin do not contain any factorXIII. The activated factor XIII can be added to the compositioncomprising noncrosslinked fibrin at a final concentration of from about1.0 to about 20 units factor XIII per ml of composition comprisingnoncrosslinked fibrin. Alternatively, the factor XIII can be supplied bythe blood or body fluids at the desired site or by the addition ofautologous plasma to the composition comprising noncrosslinked fibrin.Nonlimiting sources of calcium ions include calcium chloride, preferablyat a concentration of 30 mM. Alternatively, and less preferred, calciumions can be supplied by the blood or body fluids at the desired site.Also, as an option, thrombin can be added to such composition in orderto ensure that crosslinked fibrin II is formed. From about 4 units toabout 500 units of thrombin per ml of composition comprisingnoncrosslinked fibrin can be added or the thrombin can be provided bythe desired site.

If the noncrosslinked fibrin has been solubilized, i.e., is a fibrinmonomer, how the fibrin monomer is converted to fibrin polymer dependson how the solubilization was carried out, e.g., acid buffer orchaotropic agent. The formation of fibrin polymer can be carried out bythe same methods as described above. This fibrin polymer, if desired,can then be converted to crosslinked fibrin by the addition of a sourceof calcium ions and activated factor XIII (or precursors to activatedfactor XIII) and, optionally, thrombin to the composition comprisingfibrin monomer, as described above. The activated factor XIII can beadded to the composition comprising noncrosslinked fibrin at a finalconcentration of from about 1.0 to about 20 units factor XIII per ml ofcomposition comprising noncrosslinked fibrin. Alternatively, the factorXIII can be supplied by the blood or body fluids at the desired site orby the addition of autologous plasma to the composition comprisingnoncrosslinked fibrin. Nonlimiting sources of calcium ions includecalcium chloride, preferably at a concentration of 30 mM. Alternatively,and less preferred, calcium ions can be supplied by the blood or bodyfluids at the desired site. From about 4 units to about 500 units ofthrombin per ml. of composition comprising fibrin monomer can be addedor the thrombin can be provided by the desired site.

Fibrin Sealant Adjutants

The fibrin sealant of the subject invention can also contain adjuvants,for example, antibiotics, e.g., gentamycin, cefotaxim, nebacetin andsisomicin, histaminine H2-antagonists, e.g., ranitidine, and anticancerdrugs, e.g., OK-432. This can be carried out by adding the desiredantibiotic to the composition comprising fibrin monomer ornoncrosslinked fibrin. See M. C. H. Gersdorff and T. A. J. Robillard,Laryngoscope, 21:1278-80 (1985); A. Ederle et al., Ital. J.Gastroenterol., 21:354-56 (1991); V. Ronfard et al., Burns, 17:181-84(1991); T. Sakurai et al., J. Control. Release, 15: 39-43 1992); T.Monden et al., Cancer, 69:636-42 (1992); F. Greco, J. of BiomedicalMaterials Research, 25:39-51 (1991) and H. B. Kram et al., Journal ofSurgical Research, 50:175-178 (1991), the contents of which isincorporated herein by reference. Other adjuvants can also be added, forexample, fibronectin, fibrinolytic inhibitors such as aprotinin, alpha-2antiplasmin, PAI-1, PAI-2, 6-aminohexanoic acid, 4-aminomethylcyclohexanoic acid, collagen or keratinocytes. It is believed that thedosage of such adjuvant is the same as that utilized in conventionalfibrin sealants.

Fibrin Sealant Kits

The subject invention also provides fibrin sealant kits. The kit cancontain as a first component a composition comprising fibrin monomer anda second component an alkaline buffer that is capable of polymerizingthe fibrin monomer or distilled water, depending on how thesolubilization step was performed. The second component can optionallycontain a source of calcium ions. Alternatively, the first component canbe a composition comprising noncrosslinked fibrin and the secondcomponent can be a source of calcium ions. If the source of fibrinogenutilized to prepare a composition comprising noncrosslinked fibrin isfrom cell cultures that secrete fibrinogen or recombinant fibrinogen,the first component can be a composition comprising noncrosslinkedfibrin, the second component can be a source of calcium ions and a thirdcomponent is activated factor XIII.

EXAMPLES Plasma Preparation from Whole Blood

Whole blood (100 ml) is collected into standard commercially availableblood bag containing an anticoagulant such ascitrate/phosphate/dextrose. The blood is then transferred to a containersuitable for centrifugation and centrifuged at room temperature for 10minutes at 3,000 g. The clear supernatant plasma (approximately 50 ml)is decanted and the cellular components are discarded.

An alternative method of plasma preparation from whole blood is byfiltration. Again, 100 ml whole blood is collected into a bag containinga suitable anticoagulant such as citrate/phosphate/dextrose. The bloodis then recirculated over a filter exhibiting good protein transmissionby means of peristaltic pump. The pressure drop across the membraneresults in plasma being forced through with cellular componentsremaining in the recirculating blood. Plasma (50 ml) is collected forfurther processing.

Immobilization of Batroxobin onto Beaded Agarose

Beaded agarose (Pharmacia) was employed in immobilization studies. Theagarose used was 4% crosslinked, 45-165 μm (90% of particles). Thismaterial swells to 5 times its volume when hydrated. Batroxobin is firstoxidized with 0.1 M sodium periodate for 1 hour at room temperature in acovered scintillation vial. The batroxobin is purified by passingthrough a SEPHADEX® PD-10 gel permeation column. The oxidized batroxobinis then coupled to a hydrazide activation agarose gel overnight at roomtemperature in 50 mM sodium acetate pH 5.5 plus 10 mM sodium borohydride(30-200U per 1.75 g moist gel). The non-bound reactions are removed fromthe gel by washing with: -50 ml mM sodium acetate pH 5.5, 100 ml 50 mMglycine/1M sodium chloride pH 3.0, 100 ml 50 mM sodium carbonate/1Msodium chloride pH 10.0, 100 ml 50 mM sodium phosphate/1M sodiumchloride pH 7.0, 100 ml water, 100 ml 50 mM sodium phosphate/1M sodiumchloride pH 7.0, 100 ml 50 mM sodium phosphate pH7.0.

Reaction of Immobilized Enzyme with Plasma Fibrinogen to Form aComposition Comprising Noncrosslinked Fibrin

The immobilised enzyme (350 mg) is added to approximately 50 ml ofcentrifuged or filtered plasma and gently mixed for 7 to 20 minutesuntil a non-crosslinked fibrin I polymer clot is formed.

The fibrin I polymer plus associated immobilized enzyme is thenharvested by either: a) centrifugation at 3,000 g for 10 minutesfollowed by removal of the supernatant serum by decantation, or b)filtration through a suitable low protein binding filter followed bydiscarding the filtered serum and retaining the fibrin I polymer plusimmobilized enzyme for further treatment. This fibrin I polymer is anexample of a composition comprising noncrosslinked fibrin.

Solubilization of the Fibrin I Polymer to Form a Composition ComprisingFibrin Monomer

Solubilization of the fibrin I polymer is achieved by the addition ofabout 1-4 ml of 0.2M sodium acetate pH 4.0 containing 30 mM calciumchloride buffer to the harvested fibrin I polymer plus immobilizedenzyme. The sample is subsequently agitated vigorously, e.g., on avortex mixer, for 2 minutes. Removal of the immobilized enzyme can thenbe acheived by filtration of the fibrin I solution through a 1 to 20 μmlow protein binding filter. The agarose enzyme will be retained by thefilter, thus allowing its removal from the system. The remainingsolution, a composition comprising fibrin I monomer, consists of aconcentrated acidic fibrin I monomer solution in conjunction with otherplasma proteins. Generally, about 100 ml. of whole blood results inabout 4 ml or 5 ml of the composition comprising fibrin monomer whereinthe concentration of fibrin monomer is from about 25 mg/ml to about 35mg/ml. This is now ready for delivery to the patient either, alone or,co-extruded with repolymerizing buffer to form a fibrin sealant.

Repolymerization of the Composition Comprising Fibrin I Monomer

Repolymerization is achieved by simultaneous mixing of 9 parts of thesolution comprising fibrin I monomer with 1 part of a suitablerepolymerizing buffer, e.g., 0.75M sodium carbonate/sodium bicarbonate,pH 10.0. The ratio of mixing can be altered; however, a 9:1 ratiomaintains the high concentration of fibrin in the final product. Onco-extrusion the fibrin I spontaneously repolymerizes and converts tofibrin II followed by crosslinking of the fibrin over a period of about30 minutes.

The present invention is not to be limited in scope by the specificembodiments described, which are intended as single illustrations ofindividual aspects of the invention. Indeed, various modifications ofthe subject invention in addition to those shown and described hereinwill become apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims.

Hydrazide Activation of Agarose and Coupling VIA the Carbohydrate Moietyof the Enzyme

In order to react the carbohydrate moiety of batroxobin with a hydrazideactivated support, the sugar must first be oxidised to give free --CHOgroups. This is accomplished as follows.

Batroxobin 1-7 mg is dissolved in 2 ml of water and 0.2 ml 0.1M sodiumperiodate. The mixture is incubated at room temperature for 1 hour afterwhich it is desalted on a Sephadex® G-25 gel filtration column. Theoxidized active batroxobin is eluted from the column with 0.9% w/vsodium chloride.

Standard commercially available hydrazide activated agarose (Bio-radLaboratories Ltd UK) or any group with a terminal free amino group canbe employed to directly couple free aldehyde groups (--CHO). Ifhydrazide-agarose is used the coupling procedure is as follows.

1-5 g Hydrazide-agarose gel is suspended in 4 ml 50 mM sodium acetate pH5.5. To the suspension is added 1 ml 10 mM sodium borohydride togetherwith the desired amount of oxidized batroxobin (typically 100-200BU perg moist gel).

The sample is mixed overnight at room temperature and subsequentlywashed on a sintered glass funnel with 50 ml 50 mM sodium acetate pH5.5, 100 ml 50 mM glycine/1M sodium chloride pH 3.0, 100 ml 50 mM sodiumcarbonate/1M sodium chloride pH 10.0, 100 ml 50 mM sodium phosphate/1Msodium chloride pH 7.0, 100 ml water and 100 ml 50 mM sodiumphosphate/1M sodium chloride pH 7.0 and finally 100 ml 50 mM sodiumphosphate pH 7.0.

The method for reaction of the immobilized batroxobin with plasma is asfollows.

The pH of plasma (50 ml) is reduced to pH 5.5 by the addition of aceticacid. To the plasma is added the equivalent of 100-200BU of batroxobinimmobilized to approximately 1.75 g (moist gel weight) agarose. Theplasma is incubated at 37° C. for 10-15 minutes after which time the pHis increased to pH 7.4 by the addition of sodium hydroxide. Fibrin Ipolymerisation occurs almost instantaneously. The fibrin I polymer isharvested by centrifugation at 3,500 rpm for 10 minutes and redissolvedin 3-4 ml of 0.2M sodium acetate pH 4.0 containing 30 mM calciumchloride. Agarose-enzyme can then be separated from fibrin I bycentrifugation or filtration. The fibrin I is subsequently repolymerizedto form the fibrin sealant by the addition of 9 parts fibrin I solutionto 1 part 0.75 M sodium carbonate/bicarbonate pH 10.0.

The Enzyme Capture System using Biotin-avidin

Enzyme capture is performed by first biotinylating the batroxobin andcapturing the biotin-enzyme with immobilized avidin (avidin-coupled to6% crosslinked agarose).

Biotinylation of proteins can be acheived using a number of reagents. Inthis instance, N-hydroxysuccinimide-biotin (water insoluble) is used(commercially available from Amersham). NHS-biotin (50 μl) was added to1 mg of batroxobin at pH 8.6 and the solution is incubated at roomtemperature for 1 hour. Excess biotinylation reagent is removed by gelfiltration on a Sephadex® G-25 column with 0.1M potassium phosphate pH7.5 as the eluent.

The equivalent of 10BU (biotin-batroxobin is added to 50 ml plasma andincubated at 37° C. for 10 minutes. Fibrin I polymer is harvested bycentrifugation (3,500 rpm for 10 minutes) and redissolved in 3-4 ml of0.2M sodium acetate pH 4.0 containing 30 mM calcium chloride. To thefibrin I, is added avidin-agarose (commercially available from SigmaChemical Co.) at 0.2 g (moist gel) per ml fibrin I. The sample isincubated at room temperature for 10 minutes and centrifuged at 2,000rpm for 10 minutes. The fibrin I produced is substantially free frombatroxobin. Fibrin repolymerization to form the fibrin sealant isachieved as described in Example 5.6.

What is claimed is:
 1. A composition consisting essentially of fibrinmonomer wherein said composition is in solid form and is substantiallyfree of an enzyme which catalyzes the cleavage of fibrinopeptides Aand/or B from fibrinogen.
 2. The composition of claim 1 wherein saidcomposition is formed by lypohilizing an aqueous solution comprisingfibrin monomer.
 3. A composition consisting essentially of fibrinmonomer, selected from this group consisting of fibrin I monomer, fibrinII monomer or des-BB fibrin monomer or mixtures of any of these, in asolid form wherein said fibrin monomer is prepared by a processcomprising the steps oftreating fibrinogen with an enzyme whichcatalyzes the cleavage of fibrinopeptides A and/or B from fibrinogen toform said fibrin monomer; removing substantially all of said enzyme fromthe soformed fibrin monomer; and collecting said fibrin monomer in solidform.
 4. The fibrin monomer composition of claim 3 wherein said fibrinmonomer is collected in a solid form by the step of lyophilizing anaqueous solution of said fibrin monomer following removal of saidenzyme.